The Biosynthesis Pathway Of Sanguinarine In Macleaya Cordata Via Isotope Labeling L-tyrosine

This study has established a qualitative analyze for the rapid identification of alkaloid compounds in Macleaya cordata?Willd.?R.Br.using LC-Q/TOF MS and Personal Compound Database and Library of alkaloids.It explored the biosynthesis pathway of sanguinarine and reevaluated the reported studies of biosynthesis pathways of sanguinarine and chelerythrine by using the method of feeding ¹³C isotope-labeled precursors.Based on LC-QQQ MS/MS,a quantitative analysis method for the determination of 21 intermediate metabolites in the biosynthesis pathways of sanguinarine and chelerythrine was established,and the distribution of these intermediate metabolites in different organs was analyzed in the study.We get the following results:1.Through the LC-Q/TOF MS qualitative analysis method,a total of 740 compounds were found in different organs of Macleaya cordata,59 alkaloids were speculated by MS/MS.30 were confirmed f by comparing with standards,nineteen compounds which had been reported as intermediates in the biosynthesis pathway of sanguinarine and chelerythrine.In addition,?s?-norlaudanosoline,?s?-6-O-methylnorlaudanosoline and?s?–norlaudanosoline were first found in advanced plants.2.The quantitative analysis method was used to quantify the intermediate metabolites in the biosynthetic pathways of sanguinarine and chelerythrine by LC-QQQ MS/MS.The results showed that there were significant differences in the distribution of these intermediate metabolites in different organs,The main alkaloid is the benzophenanthridine alkaloid,sanguinarine?SAN?and chelerythrine?CHE?,and proton alkaloid,protopine?PRO?and allocryptopine?ALL?,then tetrahydroberberine alkaloids,N-methylcanadine?cp14?and N-methylstylopine?cp21?.PRO and ALL are the indirect precursors of SAN and CHE,the highest cumulative alkaloid,mainly distributed in the root content,the highest cumulative amount of close to 30 mg/g,and it was 1.5 to 2 times accumulation of flowers,leaves and fruit.The cp14 and cp21 were the direct precursors of PRO and ALL,respectively,which were mainly accumulated in flowers and fruits,with the highest levels approaching 5 mg/g.The accumulation of other intermediate metabolites is relatively low,but the distribution in different organs also has tissue specificity.The distribution of these intermediate metabolites in different organs of Macleaya cordata returned to different organs and the distribution of the transcriptional expression data of different organs and fall back to the different organs to guide the excavation of sanguinarine and chelerythrine in the synthesis of 11 functional genes of enzymatic reaction.In addition,?s?-norlaudanosoline,?s?-6-O-methylnorlaudanosoline,?s?-norreticuline were the first compounds to be quantitatively analyzed in adcanced plants.3.A stable isotope tracing method with[ring-¹³C₆]-tyrosine as a feed precursor was constructed.The intermediate metabolites of the synthesis of sanguinarine and chelerythrine could be labeled with ¹³C isotopes,The results showed that there were two new compounds of N-methylscoulerine and N-methylcheilanthantholine by isotope tracer,and it was found thattheremightbenewbypass,scoulerine—>N-methylscoulerine—>N-methylcheilanthifoline—>N-methylstylopine--->protopine.